Functional characterization of piRNPs Germ cells carry the essence of most forms of multicellular life by storing, shaping (via meiosis), and transmitting the genetic information that propagates a species. They express Piwi family proteins that bind to small RNAs known as piwi-interacting RNAs (piRNAs) to form pi-RiboNucleoProteins (piRNPs) that silence retrotransposons, a function critical for preserving genome integrity. Primary piRNAs are derived from long, single-stranded transcripts that are processed in the cytoplasmic nuage, an electron-dense structure that is in very close apposition to perinuclear mitochondria. Drosophila melanogaster expresses three Piwi proteins termed Aubergine (Aub), Piwi and Ago3. Aub-piRNAs target and cleave transposons and the piRNA response is amplified by successive rounds of Aub and Ago3 interactions, in a process known as the ping-pong cycle. Genetic studies have shown that Aub has an additional role in specification of Primordial Germ Cells (PGCs) in the embryo, along with Csul (an arginine protein methyltransferase) and Tudor. During Drosophila oogenesis, germline RNPs assemble at the posterior of the oocyte to form germ (pole) plasm. Germ plasm is transmitted to the embryo and it is necessary and sufficient to induce the formation of PGCs from undifferentiated cells. Germ plasm contains RNAs that are essential for PGC specification but the identity of many of these mRNAs and how they are anchored at the posterior of the oocyte are not known. Our laboratory discovered that Csul catalyzes symmetrical dimethylation of arginines of Piwi proteins and we showed that Aub methylation is required for germ plasm assembly, in vivo via its interaction with its receptor protein Tudor. Studies from our lab and subsequent studies from other labs have shown that the formation of RNPs that are assembled by methylated Piwi proteins bound to Tudor-domain containing proteins (Tdrds) are conserved in the germline of all animals. Our proposal will address molecular mechanisms of piRNA biogenesis, and functions of Aub-piRNPs and Aub-mRNPs in two broad Aims. In Aim 1, we will determine molecular mechanisms of piRNA biogenesis. By using multiple approaches, including HITS-CLIP and biochemical isolation of piRNA processing factors, we will examine how piRNA precursors are selected and processed into piRNAs. We will also study the role of Aub arginine methylation in quality control of piRNA processing and transposon silencing. In Aim 2, we will determine molecular mechanisms and functions of Aub piRNPs and Aub mRNPs. We will address how Piwi proteins such as Aub bind piRNAs or longer RNAs and how piRNPs bind their targets. We will identify the composition of Aub mRNPs and molecular mechanisms of how Aub germline mRNPs assemble to specify Primordial Germ Cells. We will investigate the biological significance of our findings using Drosophila genetic and transgenic approaches.